3.1.6.2 Incubations with Cloned, Expressed Enzymes Individual UGT
enzymes have been expressed in a wide variety of systems including insect cells
(Supersomes or Baculosomes),Escherichia coli, yeast, and mammalian cells.
Zakim and Dannenberg have demonstrated that the lipid membrane composi-
tion can influence activity (Zakim, 1992). There tends to be excellent protein
expression insect cells transfected with baculovirus, but when activity is
measured compared to mammalian cells systems, there appears to be significant
amounts of inactive protein due to either poor membrane insertion or improper
folding (lack of chaperones?). Bacteria do not have an ER, but alteration of the
signal sequence results in active membrane bound preparations. Yeast and
mammalian cells such as HEK293 or V79 cells have a more typical membrane
environment and may be preferable for expression of ER proteins.
In insect cells, yeast or mammalian cells, microsomal preparations can be
prepared, however, the yield of microsomal protein from cultured cells is often
low. Sonication of a frozen whole cell lysate appears to provide a fully activated
preparation and is easier than preparing microsomes. If intact microsomes are
needed from transfected mammalian cells, a recent procedure delineated by
Sukodhub and Burchell is recommended for their preparation (Sukodhub, 2005).
When comparing the activities of individual UGT enzymes for a single
substrate (isoenzyme screening), it is important to normalize for expression.
This can be done by correction for protein expression by Western blotting in
the UGT1A family with antibodies directed against the UGT1A constant
region (encoded by shared exons 2–5). However, it is not possible to correct for
protein expression when comparing between the UGT1A family and UGT2B
enzymes because the purified proteins are not available. Thus, velocity
measurements are usually based on a per milligram of protein basis, which
must be interpreted with caution.
3.1.6.3 Analytical Methods There are three basic methods used to measure
glucuronidation rates in microsomes:
(1) radiometric methods with radiolabeled substrate of^14 C-UDPGA; (2)
fluorescence disappearance with fluorescent substrates, such as 4-methylum-
belliferone; (3) chromatographic methods, most commonly HPLC-UV or
liquid chromatography–mass spectrometry LC–MS.
Radiometric methods have been widely employed for substrate screening
assays. The most common general method is to use^14 C-UDPGA as the
cosubstrate, resulting in labeled glucuronide product(s). The glucuronides are
easily separable from the^14 C-UDPGA by thin-layer chromatography (TLC) or
by high pressure liquid chromatography (HPLC). Radioactivity on TLC plates
can be counted on a plate scanner or by densitometric quantitation on film or a
phosphoimager. This method can have significantly more variability than
stand-alone HPLC or LC/MS methods. Alternatively, radiolabeled substrate
can be employed, requiring separation of the more nonpolar aglycone from the
polar glucuronide. In some cases, this can be accomplished by simple solvent
extraction, for example, with^3 H-steroids or^14 C-naphthol. Alternatively, a
58 CONJUGATIVE METABOLISM OF DRUGS